Biochemistry (Moscow), Vol. 66, No. 11, 2001, pp. 13001310. Translated from Biokhimiya, Vol. 66, No. 11, 2001, pp. 16091622. Original Russian Text Copyright © 2001 by Sineshchekov, Govorunova.

REVIEW

Rhodopsin Receptors of in Green Flagellate Algae O. A. Sineshchekov* and E. G. Govorunova

School of , Lomonosov Moscow State University, Moscow, 119899 Russia; Email: o.sineshchekov@mtunet.ru Received May 10, 2001 Revision received July 22, 2001

Abstract—Green flagellate algae are capable of the active adjustment of their swimming path according to the light direction (phototaxis). This direction is detected by a special photoreceptor apparatus consisting of the photoreceptor membrane and eyespot. Receptor photoexcitation in green flagellates triggers a cascade of rapid electrical events in the cell membrane which plays a crucial role in the chain of phototaxis and the photophobic response. The photoreceptor current is the earliest so far detectable process in this cascade. Measurement of the photoreceptor current is at present the most suit able approach to investigation of the photoreceptor pigment in green flagellate algae, since a low receptor concentration in the cell makes application of optical and biochemical methods so far impossible. A set of physiological evidences shows that the phototaxis receptor in green flagellate algae is a unique type . It shares common chromophore proper ties with from . However, the involvement of photoelectric processes in the signal transduction chain relates it to animal visual . The presence of some enzymatic components of the animal visual cascade in isolated eyespot preparations might also point to this relation. A retinalbinding protein has been identified in such preparations, the amino acid sequence of which shows a certain homology to sequences of animal visual rhodopsins. However, potential func tion of this protein as the phototaxis receptor has been questioned in recent time.

Key words: phototaxis, photoreceptor, rhodopsin, signal transduction, photoreceptor current, photoelectric processes, green flagellate algae, Chlamydomonas, Haematococcus

Motile microorganisms are capable of responding to This purely descriptive classification does not, how light stimuli by changes in their behavior, which leads to ever, take into account the diversity of signal transduction their accumulation under optimal conditions. The most mechanisms for these responses, which might involve advanced type of photoinduced behavioral responses is photodestructive processes, light energy conversion (in phototaxis, by which cells actively adjust the swimming phototrophs) or specialized photoreception [2, 3]. In path with respect to the direction of light incidence [1]. green flagellate algae photoregulatory responses of all The ability to detect this direction assumes either the three types are mediated by a specific photoreceptor sys presence of two spatially separated receptors in the cells, tem, although processes of photosynthesis take part in or (as in green flagellate algae) comparison of the illumi regulation of the phototaxis sign [4, 5]. nation of the only receptor at different orientations of the In green flagellates, photoexcitation of the recep cell in the environment—the socalled “twoinstant tor triggers a cascade of rapid electrical phenomena in mechanism”. Accumulation of the cells in the area with the cell membrane, which plays a key role in the signal optimal illumination is provided by regulation of the pho transduction chain for phototaxis and the photophobic totaxis sign, i.e., the movement toward the light source response [68]. The photoreceptor current is the earli (positive phototaxis), or away from it (negative photo est so far detected event in light regulation of behavior taxis). Besides phototaxis, more primitive behavioral in green flagellate algae. Extremely low concentration responses are known that are driven by a change in the of photoreceptor molecules in the presence of abun light intensity regardless of the light direction. Light dant photosynthetic pigments significantly complicates dependent alteration of the average swimming speed is their detection by spectroscopic techniques in vivo, as referred to as photokinesis, whereas a short stop and/or a well as its isolation and biochemical purification. This sudden change in the direction of movement in response makes electrophysiological methods the most adequate to a step light stimulus—as photophobic response [1]. of all currently available approaches not only to inves tigation of the signal transduction mechanisms for * To whom correspondence should be addressed. photoregulation of behavior in green flagellates, but

00062979/01/66111300$25.00 ©2001 MAIK “Nauka/Interperiodica” PHOTOTAXIS RECEPTORS 1301 also to in vivo studies on the photoreceptor pigments only be recorded from this part of the cell [18, 19]; more involved. over, this current is also found in “excised ”— The body of information obtained by measurement spotcontaining vesicles detached from the cells [20, 21]. of photoinduced behavioral and electric responses in Other evidences of such localization are the fixed green flagellate algae leads to the conclusion that their orientation of the chromophores in the membrane plane phototaxis receptor is a unique rhodopsintype protein. and a key role of transmembrane electric currents in sig The most direct evidence in proof of this notion is nal transduction (see below). In the eyespot region the restoration of the ability to phototax and generate pho plasma membrane and the outer envelope are toreceptor currents in blind carotenoiddeficient tightly connected, leaving, however, a free space with a Chlamydomonas mutants after the addition of exogenous constant width of 10 to 40 nm between them [17]. retinal [9, 10]. Comparative analysis of the efficiency of Electron microscopy studies revealed that this area of the various retinal isomers and analogs in experiments with plasma membrane is enriched with intramembranous such mutants established the chemical nature of the particles of 812 nm diameter [22], which might represent native chromophore of the phototaxis receptor. complexes of receptor molecules. The outer chloroplast Substantial effort has been invested in identification envelope has also been suggested as a plausible location of of genes encoding the opsin part of the photoreceptor the photoreceptor molecules in green flagellates [23, 24]. pigment in green flagellate algae, since cloning of these However, this hypothesis seems to be less likely, since the genes and their expression in model systems appears to be photoreceptor current has virtually no lagperiod [4]. the most promising (if not the only possible) means to The amount of light incident on the photoreceptor obtain preparative amounts of the photoreceptor pro periodically changes in the course of helical swimming, teins. Genes encoding retinalbinding proteins have been typical for most flagellates, when the axis of the helix is found in Chlamydomonas [11] and Volvox [12]. However, not coincident with the direction of the light. Light is at present there are doubts that these proteins have a absorbed by carotenoids accumulated in the eyespot lipid receptor function [13]. Therefore, search for receptor globules and by the chloroplast pigments, which causes opsin genes still represents one of the most urgent prob shading of the photoreceptor when the eyespot faces away lems at the present state of research into phototaxis in from the light source. Periodic alteration of the photore green flagellate algae. ceptor illumination is perceived by the cells as a signal to correct the swimming path. The mechanism of phototaxis is schematically presented in Fig. 1. The cell moves in a STRUCTURE AND FUNCTION helical path that results from the rotation of the cell OF THE PHOTORECEPTOR APPARATUS around its longitudinal axis and in the plane of flagellar beating. If the direction of the forward movement deviates A distinct photoreceptor apparatus is employed to from the light direction (13), the photoreceptor illumi track the direction of light in phototactic algae. The nation changes during the rotation cycle. It reaches its structural organization of this apparatus provides the spa maximum when the cell faces the light source with its tial anisotropy of light absorption by photoreceptor mol eyespot (2), and minimum—when the cell is in the oppo ecules. Part of this apparatus that is visible under a light site phase of the cycle (1, 3). The difference in the pho microscope is called the eyespot, or stigma, and plays a toreceptor illumination serves as the signal for the asym role of a shading device. An accessory role of the eyespot metrical motor response of the two flagella, which takes is confirmed by the phototactic ability of eyespotdefi place twice during one rotation cycle and leads to align cient mutants. ment of the swimming path with the light direction (13). In the model microorganism Chlamydomonas rein When the swimming direction becomes parallel to the hardtii, it is ~1 micron in diameter and has a lateral posi light direction as the result of corrective flagellar motions tion in the cell [14]. The eyespot position with respect to (4, 5), the photoreceptor illumination becomes constant the plane of flagellar beating is strictly defined by the during the rotation cycle, so that no more signal to structural association of the eyespot with the flagellar change the swimming path is generated. roots [15]. In Chlorophyceae, the eyespot is part of the The importance of periodical shading of the pho chloroplast and consists of one to several layers of toreceptor for phototaxis was already recognized by early carotenoid globules often subtended by thylakoid mem investigators [25, 26]. Later this notion has been comple branes [1517]. mented by a hypothesis that the eyespot in green flagellate The photoreceptor pigment is thought to be concen algae, which consists of layers with different refractive trated in the small area of cell membranes directly adja indexes, acts as a quarterwave stack enhancing the pho cent to the eyespot—in the plasma membrane or in the toreceptor illumination when the eyespot faces the light outer chloroplast envelope. Localization of the photore source [16]. This mechanism should further improve ceptor pigment in the eyespot area is most directly sup directional sensitivity of the photoreceptor apparatus ported by the finding that the photoreceptor current can compared to that resulting from shading, since the inter

BIOCHEMISTRY (Moscow) Vol. 66 No. 11 2001 1302 SINESHCHEKOV, GOVORUNOVA

Direction of light shift measured between the stimulus–response curves for the photoreceptor current corresponds to approximately 8fold attenuation of the photoreceptor illumination when the eyespot is turned away from the light source, as compared to the opposite position [28]. Some pigmentdeficient Chlamydomonas mutants have neither eyespots, nor normal . 5 Phototaxis observed in such mutants after incorporation of exogenous retinal has the opposite sign compared to the wild type. This is a direct consequence of their gener ation of maximal photoreceptor currents when being illu minated from the back side [10]. The most likely mecha nism for this phenomenon is focusing of the light by the almost transparent cell on the photoreceptor membrane, 4 i.e., “lens effect” [5].

3 SIGNAL TRANSDUCTION MECHANISMS 2

cisflagellum In green flagellate algae transduction of the light photoreceptor stimulus to the intracellular signal and its transmission to the flagellar apparatus occurs via generation of a cascade of rapid photoelectric responses of the cell membrane, transflagellum which is triggered by photoexcitation of the receptor [29, 1 30]. The responses of this cascade were initially observed by extracellular recording with a suction pipette [7, 8]. A method for photoelectric recording in cell suspensions was developed later [31] (Fig. 2). Using traditional intra cellular microelectrodes is impeded by the high sensitivi ty of these responses to a mechanical damage of the Fig. 1. Schematic representation of the mechanism for photo taxis in green flagellate algae: 15) consequent positions of the membrane [6], whereas the application of the patch cell swimming under unilateral illumination with the actinic clamp technology has not so far succeeded, because a light. See further explanation in the text. complete removal of the cell wall could not be achieved. The involvement of the extracellularly recorded pho toinduced currents in phototaxis is unambiguously con firmed by a long list of experimental evidences: spectral ference reflection dramatically decreases when the angle and light sensitivity, localization, inhibitory analysis, of light incidence deviates from the normal to the eyespot experiments with blind mutants, etc. surface. The phototactically active light induces a constant The efficiency of the modulation of the light signal flow of the current across a defined portion of the mem during helical swimming was estimated by recording pho brane in the eyespot region. After switching off the light toreceptor currents from a cell sucked into a pipette and this current decays to zero in ~50 msec, i.e., within char illuminated at different angles with respect to the eyespot acteristic time of the passive discharge of the membrane. [18]. It has been shown that the amplitude of the pho At high stimulus intensities the current kinetics compris toreceptor current is much larger in the cell facing the es an initial peak, the amplitude of which may exceed that light source with its eyespot, as compared to the cell in the of the subsequent stationary phase more than an order of opposite position. A weak dependence of the current magnitude [21, 29, 30]. The light saturation of the sta amplitude on the angle that was observed within each of tionary level is at least an order of magnitude lower than the two phases of illumination and shading of the pho that of the peak amplitude and almost corresponds to the toreceptor points to a relatively small contribution of the light saturation of phototaxis. interference mechanism to the modulation of the pho Optical monitoring of flagellar beating in a cell toreceptor illumination in Haematococcus. This conclu sucked into a micropipette revealed that a stepup light sion is consistent with the finding of only one layer of stimulus induced an increase in the beat frequency of the carotenoid globules in the eyespot of this microorganism cisflagellum (the one closest to the eyespot), and the [24]. In Chlamydomonas reinhardtii, which has the eye decrease in the beat frequency of the transflagellum, spot consisting of 24 layers of carotenoid globules [27], a whereas stepoff stimulus caused the opposite responses

BIOCHEMISTRY (Moscow) Vol. 66 No. 11 2001 PHOTOTAXIS RECEPTORS 1303 a the correction of the swimming path in a freely swimming cell, i.e., to phototaxis. Therefore, it could be concluded FC that gradual changes in the amplitude of the stationary PC photoreceptor current constitute the initial step in the flagella signal transduction chain for phototaxis (Fig. 3). 20 msec When the stimulus intensity and/or duration exceeds a certain threshold, the photoreceptor current is superim excitation posed by a transient signal of a complex waveform, ini eyespot flash tially termed as the “regenerative response” [7, 8]. This response appears as an “allornothing” event, i.e., its b amplitude only slightly depends on or does not change much upon variation of the stimulus intensity, whereas its excitation lag period becomes shorter when the intensity increases. flash The sign of its major peak measured in a cell sucked into PC a pipette depends on whether the flagella are inside or 10 msec outside the pipette, which shows that it reflects an inward PC2 PCrec transmembrane current in the flagellarbearing region of the cell [29, 33]. Therefore, this current has been referred PC1 FC to as the “flagellar current” [33]. It is highly sensitive to FC 1 excitation the removal of Ca2+ ions from the extracellular medium. flash FCrec The integral under the photoreceptor current before the onset of the flagellar current is constant at any stimulus intensity, which indicates that the flagellar current likely Fig. 2. A scheme for measurement of photoinduced electrical 2+ signals involved in the signal transduction chain for phototaxis reflects opening of voltagegated Ca channels [29, 30]. in green flagellate algae (PC, photoreceptor current; FC, fla These channels appear to be localized to the membrane gellar current): a) signal recording in individual cells with a of flagella and evenly distributed along their whole length suctionpipette technique; b) signal recording in a suspension [34]. of nonoriented cells. The index 1 shows the currents from individual cells facing the light source with their photorecep A close time correlation between the appearance of tors; the index 2, those facing away from it; the index rec, the regenerative response and a switch from ciliary style of resultant (recorded) currents. flagellar beating to undulation shows that it is the basis for the photophobic response of the cell. This notion has been recently confirmed by simultaneous measurement [18, 29, 30, 32]. The observed beat frequency changes of photoelectric currents and flagellar beating in the same were accompanied by slight changes in beating curvature, cell [35] and by photoelectric measurements from sus also opposite in the two flagella of the cell. Such unbal pensions of C. reinhardtii mutants deficient in the photo anced motor responses of the two flagella would lead to phobic response [36, 37].

asymmetric motion light ??? reaction of two cellular stimulus flagella (phototaxis)

photoexcitation of PC depolarization receptor rhodopsin generation of membrane

switch from ciliary regenerative to undulation style reaction of flagellar beating (FC generation) (photophobic reaction)

Fig. 3. A scheme for the signal transduction chain for phototaxis and the photophobic response in green flagellate algae (PC, photorecep tor current; FC, flagellar current).

BIOCHEMISTRY (Moscow) Vol. 66 No. 11 2001 1304 SINESHCHEKOV, GOVORUNOVA The onset of the photoreceptor current measured in the data assuming a contribution of a second lowsaturat single cells of Haematococcus pluvialis upon laser flash ing process [29]. excitation is observed instantaneously within the time res Two phases of the stimulus–response curve are most olution of the measuring system (<30 µsec) [38]. clearly seen when the currents are measured in cell sus However, analysis of the signal rise shows the presence of pensions, since in this case measurements can be extend a current component with a lag period of several hundreds ed to lower light intensities due to a high signaltonoise of microseconds, the duration of which depends on the ratio [31]. Fitting of the curve is only possible with two stimulus intensity [38]. Switching on the red background saturation functions (Fig. 4). The ratio between the illumination known to hyperpolarize the cell membrane amplitudes of the low and highsaturating components [6], only increases the amplitude of the delayed compo varies from 1 : 10 to 1 : 5, and the ratio between their sat nent of the photoreceptor current [38]. The two compo uration levels varies from 1 : 300 to 1 : 50 in different nents distinguishable in the current rise are referred to as species and culture states. the “early” and “late” photoreceptor currents [4, 29, 30]. One possible explanation for the low light saturation The flashtopeak time of the photoreceptor current of the first phase of the stimulus–response curve is its shortens with the increase in the stimulus intensity [31]. being limited by the concentration of a secondary mes Its decay can be deconvoluted by at least two exponential senger. Therefore, a hypothesis has been suggested that components, although they cannot be directly related to the lowsaturating phase of the curve reflects saturation of the components of the signal rise [38]. the late photoreceptor current, whereas the highsaturat The presence of a lightdependent delay and the ing phase is determined by the saturation of the early pho sensitivity of the current amplitude to the physiological toreceptor current [4]. However, this hypothesis cannot state of the cell indicates the likely involvement of bio explain the high sensitivity of the current amplitude to the chemical mechanisms in generation of the late photore physiological state of the cell at high stimulus intensities, ceptor current. Several enzymes characteristic for senso so that the interpretation of the two phases of the stimu ry transduction cascades in animals have been detected lus–response curve at present remains unclear. in isolated eyespot preparations of green flagellate algae The causal relationship between the two receptor (see below), although their possible role in phototaxis currents in green flagellate algae is also not yet known. signaling has yet to be elucidated. On the contrary, the Since most physiological evidences point to a single absence of a lag period for the early receptor current rhodopsin species to mediate both phototaxis and the shows that it is maintained by the rhodopsin itself, or by photophobic response, it is likely that this species is a closely associated ion channel. Fast signals reflecting responsible for both currents that develop in parallel or in intramolecular processes are recorded, for instance, series. However, a possibility of the two currents being from purple membranes that contain mediated by different rhodopsins cannot be totally ruled [39]. An electrical signal with similar properties, also out [44]. called the “early photoreceptor current”, is known in animal visual cells, where it can be directly correlated to the photochemical conversion of the visual rhodopsin monitored by optical methods [40, 41]. In contrast to the currents in animal visual cells, the early receptor current in green flagellate algae is significantly overlapped by the late receptor current. This feature and the present 4 highsaturating absence of spectroscopy data on algal photoreceptor pig phase ments significantly complicate investigation of the early receptor current in flagellates. Therefore, it is difficult to estimate how similar is the origin of the early receptor 2 current in green flagellate algae to that in animal visual lowsaturating cells, where several processes, such as chromophore PC amplitude, nA phase dipole orientation, fixed charge separation, macrodipole (α helix) movement, intramolecular proton transfer, and vectorial proton uptake or release from the interfacial 0 membrane boundary solutions, have been proposed to 0.001 0.01 0.1 1 10 100 contribute to it [42, 43]. Measurement of the dependence of the peak ampli Flash intensity, relative units tude of the photoreceptor current in green flagellates on Fig. 4. Dependence of the amplitude of the photoreceptor cur the stimulus intensity also reveals its complex nature. It rent (PC) recorded in suspensions of Chlamydomonas rein was already found in experiments in individual cells that hardtii on the intensity of the excitation flash. Solid line is the an exponential saturation function can only be fitted to result of a computer fit with a sum of two hyperbolic functions.

BIOCHEMISTRY (Moscow) Vol. 66 No. 11 2001 PHOTOTAXIS RECEPTORS 1305 Molecular components of the enzymatic signaling taining flavins or carotenoids as their chromophores. cascade that is characteristic for visual receptor cells have Lack of a near UV band in phototaxis spectra in the above been identified by biochemical methods in the eyespot microorganisms ruled out the former possibility [58]. preparations from green flagellate algae. The isolated Coincidence of the overall shape of these spectra and that Chlamydomonas eyespots have been reported to in vitro of the absorption spectrum of animal visual rhodopsin led mediate the lightdependent activity of cGMPphospho to the hypothesis that phototaxis receptors in green flag diesterase from bovine in the presence of bovine ellate algae might be homologous to the pigments of ani transducin [45, 46]. Membranes of a carotenoiddeficient mal vision [16]. Chlamydomonas mutant did not have this capacity, unless Theoretical considerations imply that the action they were preincubated with 11cis or alltransretinal spectrum of phototaxis should result from a superposition [47]. The membranes, preincubated with either retinal of the absorption spectrum of the receptor pigment and isomer, but not untreated, also catalyzed photostimula the spectral characteristics of the eyespot and chloroplast, tion of transducin GTPase [47]. These findings have been which modulate the photoreceptor illumination during interpreted as an indication to that Chlamydomonas pho phototaxis. This notion has been experimentally con totaxis receptor has the binding site for animal transducin firmed [56, 59, 60]. The action spectrum that provides the [46]. closest match of the absorption spectrum of the photore Several proteins with characteristics of subunits of ceptor pigment was obtained by measurement of the pho heteromeric G proteins were detected in the eyespot toreceptor current at maximal illumination of the pho preparations from the green flagellate algae C. reinhardtii toreceptor [7, 8, 10]. Comparison of this action spectrum and S. similis [4850]. The GTPase activity measured in with that of phototaxis revealed that the contribution of the isolated eyespots of S. similis is regulated by light and the eyespot and chloroplast (except the longwavelength Ca2+ [51]. The antibodies raised against the retinalbind area of the latter) did not significantly shift the phototaxis ing protein identified in the eyespots of C. reinhardtii [52] action spectrum from the absorption spectrum of the recognized a specific 32 kD protein in the eyespot prepa receptor pigment. rations of S. similis and inhibited light modulation of their Investigation of the photoreceptor current, which is GTPase activity [51]. In addition, Ca2+dependent pro the earliest detectable event in the signal transduction tein kinase and phosphatase activities were found in these chain of phototaxis, enabled the resolution of character preparations [50, 53]. istics of the photoreceptor pigment that is not presently These data led to a hypothesis that heteromeric available for application of spectroscopic and biochemi GTPases are involved in the signal transduction chain in cal methods. An estimate of the product of the optical green flagellate algae, which is mostly supported by an cross section and quantum efficiency calculated from observation that the light dependence and spectral sensi measuring the stimulus–response dependence of the peak tivity of the photoinduced inhibition of the GTPase activ amplitude of the photoreceptor current is approximately ity correlate to those of phototaxis [51]. However, it is dif 8·10–21 m2, which is close to that of other known retinal ficult to establish whether this correlation is specific due proteins [29, 30]. Besides providing indirect evidence for to a large amount of carotenoids and other pigments with the rhodopsin nature of the pigment, this result rules out spectral characteristics close to those of the receptor the involvement of a lightharvesting (“antenna”) com rhodopsin found in the eyespot preparations. Besides plex in the phototaxis receptor. that, it is not known to what extent results obtained in Maximal amplitude of the photoreceptor current was isolated eyespot preparations reflect processes in intact recorded when the plane of polarization of the light stim cells rather than artifacts related to cell disruption. ulus was parallel to the photoreceptor membrane [18, 30, 61]. This result shows that transition dipole moments of chromophore molecules lie in the plane of the cell mem THE PHOTOTAXIS RECEPTOR IS A RETINYLI brane, which is typical for animal rhodopsins [62] and DENE PROTEIN bacteriorhodopsin [63]. Specific inhibition of phototaxis and the photore Initial ideas on the chemical nature of receptors for ceptor current in green flagellate algae Chlamydomonas photobehavioral responses were gained by measurement and Haematococcus by hydroxylamine, an agent known to of their spectral sensitivity. Action spectra with maxima induce lightdependent cleavage of the chromophore in between 450 and 500 nm have been reported for photo retinalcontaining proteins, i.e., those in which retinal taxis and the photophobic response in a number of binds to the apoprotein via formation of a Schiff base, Chlorophyta (Platymonas subcordiformis, Dunaliella sali further proved the rhodopsin nature of the phototaxis na, Stephanoptera gracilis, gametes of Ulva taeniata and receptor [64, 65]. But, the most unambiguous evidence Ulva rigida [54]; Chlamydomonas reinhardtii [55]; Volvox for such nature has been obtained by reconstitution stud aureus [56]), as well as in the dynoflagellate Gymnodinium ies in a carotenoiddeficient mutant of C. reinhardtii [9]. splendens [57]. Such spectra point to the pigments con Such mutants are blind, i.e., their phototaxis sensitivity is

BIOCHEMISTRY (Moscow) Vol. 66 No. 11 2001 1306 SINESHCHEKOV, GOVORUNOVA several orders of magnitude below that of the wild type. CHROMOPHORE PROPERTIES However, it can be restored by the addition of exogenous retinal [9]. No photoinduced electrical currents could be Investigation of the recovery of phototactic ability in detected in blind mutants unless they were reconstituted blind Chlamydomonas mutants upon the addition of with retinal [10]. This indicated that restoration of their exogenous compounds [9] turned out to be an excellent phototaxis sensitivity after the addition of retinal indeed experimental system for elucidation of chromophore resulted from reconstitution of the functional receptor requirements of algal rhodopsins. However, use of this from a normal apoprotein, expressed in these mutants, assay resulted in a major controversy between the results with the exogenous chromophore rather than from the of initial experiments carried out in the FN68 strain of C. influence of retinal on downstream elements of the signal reinhardtii by measuring the lightinduced migration of a transduction chain or its incorporation into the eyespot. cell population in a Petri dish [73, 74] and subsequent These data strongly support the notion that the native studies that employed more technically advanced meth phototaxis receptor in Chlamydomonas is a retinylidene ods. protein. Initial conclusions drawn from measurements of the C. reinhardtii is the only so far studied flagellate population migration were that the photoreceptor in species where carotenoiddeficient mutants are avail Chlamydomonas incorporates 11cisretinal and is homol able, which made it possible to carry out retinal recon ogous to animal rhodopsins [9]. Furthermore, it was also stitution studies to directly prove the rhodopsin nature concluded that its photoactivation does not involve cis of the phototaxis receptor. Inhibition of phototaxis in transisomerization of the chromophore [7274]. Haematococcus lacustris observed when the cells were However, these notions could not be confirmed by video cultivated in the presence of the carotenoid biosynthe recording and motion analysis of individual cell tracks in sis inhibitor norflurazon [66], does not necessarily the CC2359 blind Chlamydomonas strain [75, 76]. reflects the loss of the functional photoreceptor, but Neither phototaxis, nor the photophobic response might be explained by deterioration of the eyespot by assayed by this method could be restored by the addition this treatment. However, the involvement of rhodopsin of retinal analogs prevented from isomerization around type receptors in phototaxis and the photophobic the C13=C14 double bond (locked in either the 13trans response can at present be postulated for a much wider or 13cisconfiguration). Moreover, these analogs were range of flagellate species, in the first place those that inefficient in restoration of photoreceptor currents in possess intrachloroplast eyespots. This view is strongly blind mutants, although entered the retinal binding site, supported by measurement of typical photoreceptor as it could be concluded from their inhibition of the currents in a number of flagellate algae, including response induced by alltransretinal [10] (Fig. 5). Chlamydomonas, Haematococcus, Polytomella, Sperma Furthermore, it was alltransretinal which induced most tozopsis, Hafniomonas [17, 67] and Volvox [21], i.e., in rapid and most complete reconstitution of both photo most of the objects with rhodopsinlike phototaxis taxis and the photophobic response compared to its cis action spectra. isomers [75, 76]. Similar results were obtained when pho Two major groups of so far known retinal proteins tobehavioral responses were monitored by recording light comprise animal visual rhodopsins and proteins respon scattering transients in cell suspensions [77]. sible for photoreception and energy conversion in Taking into account all these observations, it was archaea [68]. Is is noteworthy that there is practically no concluded that alltransretinal is the native chro homology between primary sequences of the proteins mophore of Chlamydomonas rhodopsin(s) responsible for that belong to the different groups. Besides this, the pro both phototaxis and the photophobic response [78, 79]. teins from the two groups contain different retinal iso Photoexcitation of the receptor gives rise to isomerization mers as chromophores and undergo different primary of the chromophore to a 13cisform, as it takes place in reactions upon photoexcitation. In archaeal rhodopsins all so far known archaealtype rhodopsins. Common fea alltransretinal chromophore photoisomerizes into 13 tures of the chromophore of Chlamydomonas photorecep cis, whereas 11cisretinal is the most frequently found tor and those of archaeal rhodopsins revealed by reconsti chromophore in animal visual rhodopsins, which con tution studies in blind mutants also include its 6strans verts into alltransretinal during the photocycle. A conformation [76, 80]. Analysis of the efficiency of vari recent finding of a archaealtype rhodopsin in fungi [69, ous ring/chain conformers in restoration of the photo 70] and eubacteria [71] shows that proteins of this type phobic response in Chlamydomonas reinhardtii and are widely spread among representatives of evolutionari Halobacterium salinarium, and the results of the optical ly very distant taxa. Taking this into account, identifica studies on reconstituted archaeal rhodopsins led to the tion of what particular retinal isomer serves as the chro conclusion that the retinal binding site of Chlamydomonas mophore in phototaxis receptors in green flagellate algae photoreceptor is phylogenetically related to that of the appears to be extremely important for characterization of sensory rhodopsin II [80]. The functional chromophore these pigments. of Chlamydomonas rhodopsin requires the presence of at

BIOCHEMISTRY (Moscow) Vol. 66 No. 11 2001 PHOTOTAXIS RECEPTORS 1307 A tion, which might be enough to induce the synthesis of endogenous retinal [82]. a In vitro approaches to identification of the chro mophore of algal rhodopsins included high performance 2 liquid chromatography (HPLC) analysis of organic extracts and optical spectroscopy studies on isolated pho b toreceptor preparations. Differential centrifugation of subcellular material obtained by disruption of cells allows separation of the eyespot fraction. This fraction can be c 1 recognized as a highly pigmented orangecolored band 200 pA due to the presence of carotenoid globules [83, 84]. Electron microscopy revealed that the isolated eyespots 10 msec light retain the leaflet of the plasma membrane where the pho impulse toreceptor molecules are presumably located [84]. In Chlamydomonas, alltransretinal was the major isomer B detected in such preparations, although a small amount of 13cis, but not 11cis, retinal was also found [83, 85]. However, both alltrans and 11cisisomers were extract ed from the eyespot preparations of Spermatozopsis [86]. 0.8 As it has been already mentioned, phototactic sensi tivity in the carotenoiddeficient FN68 Chlamydomonas strain can be restored by irradiation with green light, 0.4 which presumably activated retinal biosynthesis [82]. The 1 amount of alltransretinal extractable from these cells during the irradiation increased in parallel with the 2 0.0

PC sensitivity, relative units relative PC sensitivity, increase in phototactic sensitivity, and reached ~30,000 molecules per cell [83], which is close to an estimate of 400 450 500 550 the number of receptor molecules calculated from the Wavelength, nm threshold sensitivity of phototaxis [16]. This observation indicates that the extractable alltransretinal serves as the chromophore of the phototaxis receptor. Fig. 5. A) Photoelectric signals measured in suspensions of the Detection of the rhodopsin by absorption spec carotenoiddeficient strain CC2359 of Chlamydomonas rein troscopy in isolated eyespots is very much compromised hardtii after the addition of retinal (1), its 13translocked ana by both high content of carotenoids still found in these log (2) or their combination: a) cells were preincubated with 15 nM analog; b) the same, but 10 nM retinal was added afterwards; c) preparations even after detergent treatment, and by their cells were preincubated with 10 nM retinal. B) Action spectra intense light scattering due to the presence of membrane for the photoreceptor current measured in suspensions of a fragments. Absorption changes observed after bleaching carotenoiddeficient mutant after the addition of 5 nM retinal of the isolated eyespots with green light seem to be most (1) and the wild type of Chlamydomonas reinhardtii (2). ly driven by oxidation of carotenoids, since it was signifi cantly reduced in the presence of the antioxidant αtoco pherol (E. Govorunova, unpublished observations). The very little effect of hydroxylamine on the bleaching process [83] also supports this view. The addition of least three conjugated C=C double bonds in the polyene exogenous retinal has been reported to restore the absorp chain and a methyl group at C13 position, as it has been tion of the bleached eyespot preparations, although dif found by a behavioral assay [77] and by recording photo ferent retinal isomers were found to be the most efficient electric currents in cell suspensions [10]. in different studies [83, 86, 87]. This discrepancy likely The discrepancies between the results of reconstitu indicates that the enrichment of the receptor content in tion experiments in “blind” Chlamydomonas cells report the eyespots preparations is insufficient for the applica ed by Foster’s group and those of subsequent studies tion of optical spectroscopy methods. might have several reasons. These are using in the former case high chromophore concentrations that might con tain trace retinal impurities sufficient to restore the pho THE SEARCH FOR OPSIN GENES toreceptor function, and that might accumulate in the eyespot lipid globules [81], and also using extended (10 min) Alltransretinal chromophore of phototaxis recep illumination for measurement of the population migra tors of green flagellate algae points to their similarity with

BIOCHEMISTRY (Moscow) Vol. 66 No. 11 2001 1308 SINESHCHEKOV, GOVORUNOVA archaeal rhodopsins. However, all attempts to identify opsins, but not to the opsins from archaea, and contains genes homologous to those of archaeal rhodopsins which many polar and charged residues, as is typical for ion have been undertaken by several research groups, were, to channels [11]. The chlamyopsin gene could be heterolo the best of our knowledge, so far unsuccessful. gously expressed with a high yield in Escherichia coli, On the other hand, a homologous fragment was Schizosaccharomyces pombe, and Pichia pastoris, but no detected in DNA isolated from Chlamydomonas using a retinal binding was observed in the resulting protein (W. bovine rhodopsin complementary DNA probe for Deininger, unpublished observations). A synthetic hybridization [88]. However, this result does not neces codonadapted gene encoding green fluorescent protein sarily imply that this fragment encodes the phototaxis (GFP) was fused to the chlamyopsin gene and expressed receptor protein rather than any other Gproteincou in C. reinhardtii [89]. Although the expression level of the pled membrane receptor which might also be present in chlamyopsinGFP was low, fluorescence microscopy unicellular algae. studies have shown that the fusion protein was localized Insertional mutagenesis has been used to identify to the eyespot region of the cell [89]. genes that encode molecular components of the photo Screening of the cDNA library of the colonial alga taxis signaling pathway in Chlamydomonas, and 12 ptx Volvox carteri with fragments of the chlamyopsin cDNA mutations that cause defects in phototaxis were identified resulted in identification of a clone carrying a cDNA by this approach [36]. However, most of these mutants insert encoding a protein with the deduced amino acid displayed normal photoreceptor currents, thus bearing a sequence 61% identical to that of chlamyopsin [12]. This deficiency in the signaling cascade downstream from the protein, named volvoxopsin, was expressed in S. pombe, photoreceptor. In those cases when inhibition of the pho and the antibodies raised against it were used to study its toreceptor current was observed, its detailed investigation expression during the lifecycle of Volvox. Surprisingly, the revealed that this inhibition was not due to disruption of volvoxopsin concentration was found to be higher in the the receptor protein, but rather due to indirect reasons. In total membrane fraction of gonidia, which lack visible particular, a decreased amplitude of the photoreceptor eyespots, than in the total membrane fraction of somatic current recorded by a population assay in the ptx4 mutant cells [12]. is the consequence of its multiple eyespots. A twofold All attempts to produce knockout mutants for opsin reduction of the photoreceptor current in the ptx3 mutant genes either in Chlamydomonas or Volvox were so far is not enough to explain its large defect in phototaxis, so unsuccessful, which significantly complicates functional that the current appears to be indirectly affected by this testing of the products of these genes as possible candi mutation. Therefore, no mutants that have the phenotype dates for phototaxis receptors. However, in both microor expected for the gene encoding the photoreceptor protein ganisms, reduction of the opsin concentration could be have been so far obtained by this approach [36]. achieved by transformation with antisense constructs [12, An alternative strategy for identification of a photo 13]. In Volvox, a decreased light sensitivity of photoaccu taxis receptor gene assumed a protein as the starting mulation in the opsin antisense transformant was report point. A single protein was detected by labeling with 3H ed [12]. However, the analysis of the opsin antisense containing retinal in the eyespot membrane fraction from transformants produced from three different strains of the wild type Chlamydomonas cells, which constituted Chlamydomonas revealed no difference in the light sensi about 25% of total protein in this preparation, but was tivity of their photoreceptor currents, phototactic orien almost absent in other cell membranes [83]. tation and the photophobic response, as compared to Preincubation of the membranes with 9azidoretinal, their parental strains [13]. Therefore, the functional role which, according to the results of physiological reconsti of the product of this gene identified in Chlamydomonas tution studies, cannot enter the retinal binding site due to remains unclear, but its being the phototaxis receptor its bulky substituent, improved labeling with 3Hcontain seems to be unlikely. ing retinal likely due to saturation of nonspecific binding Today we can state that substantial efforts invested in of the chromophore [52]. This protein has been purified studies on phototaxis in green flagellate algae since the to homogeneity by SDSPAGE and used to raise a poly discovery of this phenomenon at the end of the XIX cen clonal antibody [11]. Immunofluorescence experiments tury succeeded in a rather clear outline of the orientation have shown that the antibody concentrates in a small mechanism and basic components of the signaling cas clearly defined area recognized as the eyespot in partially cade. There are also no doubts that rhodopsintype pro permeabilized cells, although direct observation of the teins serve as phototaxis receptors in this group of eyespot position in such cells was not possible [11]. microorganisms, although their unambiguous identifica Peptides derived from the purified protein were used tion has not been achieved yet. Their properties revealed for mRNA isolation and preparation of cDNA that con by indirect methods indicate that these pigments consti tained an open reading frame encoding a protein of 235 tute a unique group of retinalbinding proteins. amino acids [11]. This amino acid sequence, referred to On one hand, they share the same chromophore type as chlamyopsin, shows some homology to invertebrate with archaeal rhodopsins. Identification of genes, homol

BIOCHEMISTRY (Moscow) Vol. 66 No. 11 2001 PHOTOTAXIS RECEPTORS 1309 ogous to those of archaeal rhodopsins, in some eukaryot 13. Fuhrmann, M., Stahlberg, A., Govorunova, E., Rank, S., ic microorganisms shows a possibility of the presence of and Hegemann, P. (2001) J. Cell Sci., 114, 38573863. such genes also in green flagellate algae, although they 14. Rueffer, U., and Nultsch, W. (1985) Cell Motil. have not yet been found there. On the other hand, the Cytoskeleton, 5, 251263. 15. Melkonian, M., and Robenek, H. (1984) involvement of photoelectric processes in the signal Prog. Phycol. Res., 3, 193268. transduction chain is a common feature of phototaxis 16. Foster, K.W., and Smyth, R. D. (1980) Microbiol. Rev., 44, receptors and animal visual rhodopsins. The presence of 572630. several components of the visual enzymatic cascade in the 17. Kreimer, G. (1994) Int. Rev. Cytol., 148, 229310. eyespot preparations further supports the similarity 18. Sineshchekov, O. A. (1988) in Phototrophic Microorganisms between algal and animal signaling systems. At the same (Gogotov, I. N., ed.) [in Russian], AN SSSR, Pushchino, time, the photoreceptor function of the retinalbinding pp. 1118. proteins found in Chlamydomonas and Volvox, whose pri 19. Ristori, T., Ascoli, C., Banchetti, R., Parrini, P., and mary sequences show a homology to animal rhodopsins, Petracchi, D. (1981) in Proc. 6th Int. Congr. Protozool., Warsaw, p. 314. at present cannot be considered proven. 20. Holland, E.M., Braun, F.J., Nonnengaesser, C., Harz, Therefore, it can be concluded that further investiga H., and Hegemann, P. (1996) Biophys. J., 70, 924931. tion of the properties of the unique rhodopsin receptors 21. Braun, F.J., and Hegemann, P. (1999) Biophys. J., 76, for phototaxis in green flagellate algae and, in the first 16681678. place, identification of the genes encoding for them, will 22. Melkonian, M., and Robenek, H. (1980) J. 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